Human cytomegalovirus (HCMV) is a significant human pathogen that infects the majority of the world's population, causes birth defects and severe disease in patients with suppressed immune function, and is associated with immunosenescence and proliferative diseases such as cancer, restenosis, and transplant-associated vasculopathy. Both primary infection and reactivations of latent infections cause HCMV disease. The HCMV pp71 protein is an attractive target for antiviral therapy because it is a transcription factor and cell cycle regulator that performs critical functions at the very beginning of both lytic and latent infections. Our long- term goal is to determine the molecular mechanisms behind each function of pp71 in HCMV-infected cells, the contribution that the different activities of pp71 make during lytic and latent viral replication cycles, and to use this knowledge to design antiviral strategies against HCMV that target the pp71 protein. pp71 is a tegument protein that is delivered to cells by infectious particles and controls the expression of the viral immediate early (IE) genes. IE gene expression initiates lytic replication, and must be silenced in order for the virus to establish latency. Our published work shows that a cellular protein called Daxx silences HCMV IE gene expression, and that during a lytic infection, tegument-delivered pp71 localizes to the nucleus and degrades Daxx in a proteasome-dependent, ubiquitin-independent manner to stimulate IE gene expression (63,132). Our new preliminary data indicates that pp71 is necessary and sufficient to induce a modified form of Daxx. Because the timing of this modification correlates with Daxx degradation, we will test if this modification of Daxx is the ubiquitin-independent signal for Daxx degradation. We have also recently published that tegument-delivered pp71 is trapped in the cytoplasm and thus fails to degrade Daxx in cells where HCMV establishes a quiescent or latent-like infection in vitro (133). In these cells, IE genes are silenced in a Daxx-dependent manner. Thus, Daxx stability upon HCMV infection determines whether infected cells initiate lytic, or establish latent- like infections. Our new preliminary evidence indicates that Daxx and pp71 also control true latent infections established in CD34+ undifferentiated myeloid cells. Because CD34+ cells are difficult to maintain as undifferentiated cells in culture, we are developing embryonic stem cells as a more versatile model to study HCMV latency. Here we propose to further our examination of the role that pp71-mediated degradation of Daxx plays during HCMV latency, and to define the molecular mechanism through which pp71 degrades Daxx. This proposal builds upon our published and unpublished discoveries, utilizes state of the art molecular and genetic approaches, and introduces creative and innovative new methodologies to study HCMV latency. PUBLIC HEALTH RELEVANCE: Almost everyone is infected with human cytomegalovirus (HCMV), and once infected, can never get rid of the virus. HCMV causes disease in young, old, sick, and otherwise healthy patients. We are studying how the virus begins to replicate in the hope of finding ways to stop viral replication and thus treat HCMV disease.